Cyclosporin A is a potent immunosuppressive agent which has been successful in prolonging the survival of kidney, liver and heart allogeneic transplants in humans. Cyclosporin A has been demonstrated to suppress some humoral immunity and, to a greater extent, cell-mediated reactions such as allograft rejection and delayed hypersensitivity. Other cyclosporins, such as cyclosporin G, have also been used as immunosuppressive agents, but are not as widely used as cyclosporin A. High doses of cyclosporin A can cause hepatotoxicity and nephrotoxicity and low doses can leas to possible organ rejection. For these reasons, patients receiving cyclosporin A should be monitored at repeated intervals for cyclosporin A blood levels and subsequent dose adjustments should be made.
In blood, cyclosporin A partitions into red blood cells. Partitioning of cyclosporin A increases as the temperature of blood decreases from body temperature to room temperature. Therefore, to eliminate analytical variability due to time and temperature equilibration, it is advantageous to perform determinations of cyclosporin A blood levels on whole blood samples. Currently, cyclosporin A levels in whole blood samples are determined using radioimmunoassays and high performance liquid chromatography (HPLC). Radioimmunoassays are less advantageous than other immunoassays, such as enzyme-linked immunoassays, because radioimmunoassays require the use of a radioisotope which poses numerous problems associated with handling, storage, and disposal. HPLC is less advantageous than other assays because it requires laborious procedures, such as solvent extraction of cyclosporin A. There have been no cyclosporin A non-radiometric immunoassays described which can be performed directly on whole blood samples.
Enzyme-linked immunoassays have achieved widespread use for the measurement of clinically important analytes. There are many different types of enzyme-linked immunoassays, such as sandwich immunoassays and competitive and noncompetitive heterogeneous immunoassays. Enzyme-linked immunoassays often utilize an enzyme-labeled antibody specific for the analyte of interest. the enzyme-labeled antibody binds to an analyte of interest in a sample and the enzymatic activity of either the bound enzyme-labeled antibody or the free enzyme-labeled antibody is measured by reacting the enzyme with a substrate to produce a detectable chromophore. Affinity columns containing immobilized analyte are often used to separate the free labeled antibody from the bound labeled antibody so that the enzymatic activity of the bound labeled antibody can be measured. Virtually any enzyme that can be coupled to an antibody and can react with a substrate to produce a detectable chromophore can be used in enzyme-linked immunoassays.
An enzyme-linked immunoassay which is to be performed on whole blood samples has unique requirements for selection of enzyme label and enzyme substrate. Since a whole blood sample can contain lysed red blood cells, an enzyme label used in an enzyme-linked immunoassay performed on whole blood samples cannot be endogenous to red blood cells. The enzyme .beta.-D-galactosidase is not endogenous to red blood cells and is a suitable enzyme label for an enzyme-linked immunoassay performed on whole blood samples.
Red blood cells contain heme, which has an intense absorption peak at 405 nm, known as the Soret band. The absorbance peak at 405 nm is so intense that the measurement of absorbance of a 10 .mu.L whole blood sample diluted 500-fold would be beyond the capacity of a conventional spectrophotometer. Therefore, a suitable .beta.-D-galactosidase substrate for use in an enzyme-linked immunoassay performed on whole blood samples should produce a chromophore which does not absorb at or near 405 nm since the interference from the Soret band would be too great. A .beta.-D-galactosidase substrate most commonly used in enzyme-linked immunoassays is o-nitrophenyl-.beta.-D-galactopyranoside (ONPG). However, since ONPG produces a chromophore which absorbs at 405 nm, it is not a suitable .beta.-D-galactosidase substrate for use in an enzyme-linked immunoassay performed on whole blood samples.
Two classes of .beta.-D-galactosidase substrates producing chromophores which absorb in the visible range, approximately 560 nm to 590 nm, are phenolsulphonphthaleinyl-.beta.-D-galactosides (U.S. Pat. No. 4,668,622, issued May 26, 1987, to Kuhr et al.) and glycosides of resorufin derivatives (DE 3411574, issued Oct. 3, 1985). These substrates are described as being useful for enzyme immunoassays in which .beta.-D-galactosidase is used as an indicator enzyme. While no interference in absorbance measurement would be caused by the Soret band, oxyhemoglobin, another consitituent of red blood cells, has an absorbance peak at 577 nm. Therefore, the two above-identified classes of .beta.-D-galactosidase substrates are not expected to be suitable for an enzyme-linked immunoassay performed on whole blood samples because of interference from oxyhemoglobin.
There remains a need for an enzyme-linked immunoassay for the direct and rapid measurement of cyclosporin A levels in whole blood samples.